Possible KIR-driven genetic pressure on the genesis and maintenance of specific HLA-A,B haplotypes as functional genetic blocks.

The HLA genomic structure underlines the permanence of fixed haplotypes transmitted in blocks as allelic combinations. One of the most discussed concerns is how and why such a strong linkage between HLA alleles has been maintained for so long. We hypothesized a possible KIR-driven pressure in the genesis of specific HLA-A,B haplotypes. Certain HLA-A and -B molecules are ligands for the same KIR receptors through the Bw4 binding motif spanning residues 77-83 in the α1 domain. We analyzed the HLA-A and -B genomic types of 9897 Caucasian people (3533 newborns and 6364 adults) subdividing them according to the presence/absence of the HLA-B Bw4 serological epitope. For each HLA-B Bw4- and Bw6-cross-reactive group, we evaluated the presence/absence of HLA-A ligands for KIR3DL1 (HLA-A*23, HLA-A*24, HLA-A*32) and KIR3DL2 (HLA-A*03, HLA-A*11). The frequency of HLA-A KIR ligands significantly increased moving from the HLA-B Bw4/Bw4 to the HLA-B Bw4/Bw6 and the HLA-B Bw6/Bw6 groups among both newborns and adults (P<0.0001). Here, we suggest that, when the HLA-B KIR-ligand motif is lacking, the HLA-A KIR-ligand might have a vicarious role in controlling the natural killer cell-mediated innate immune response. Basing upon this compensatory function in the engagement of KIR receptors, we hypothesize that specific HLA-A,B ancestral haplotypes were generated.

Cytokine genetic profile in Whipple's disease.

Whipple's disease (WD) is a very rare chronic systemic condition characterised by a Th2/T regulatory (Treg) dysregulated immune response versus Tropheryma whipplei, a bacterium widely diffuse in the environment. To investigate whether this Th2/Treg polarised response has a genetic background, we investigated the Th1, Th2, Th17 and Treg cytokine genetic profile of 133 patients with WD. Thanks to the European Consortium on WD (QLG1-CT-2002-01049), the polymorphism of 13 cytokine genes was analysed in 111 German and 22 Italian patients using the polymerase chain reaction with sequence-specific primers (PCR-SSP) technique. The frequencies of the genotypes, haplotypes and functional phenotypes were compared with those obtained in 201 German and 140 Italian controls. Clinical heterogeneity was also considered. Functionally, WD patients may be considered as low producers of TGF-ß1, having an increased frequency of the genotype TGF-ß1+869C/C,+915C/C [12.3 % vs. 3.81 %, odds ratio (OR) = 4.131, p = 0.0002] and high secretors of IL-4, carrying the genotype IL-4-590T/T (5.34 % vs. 1.17 %, OR = 5.09, p = 0.0096). No significant association was found between cytokine polymorphism and clinical variability. Analogously to the recent cellular findings of a Th2/Treg polarised response, we showed that the cytokine genetic profile of WD patients is skewed toward a Th2 and Treg response. This was similar in both German and Italian populations. However, the significant deviations versus the controls are poorer than that expected on the basis of these recent cellular findings.

HLA haplotypes and birth weight variation: is your future going to be light or heavy?

Birth weight is known to be a direct indicator of perinatal mortality and a clear predictor of adult pathologies too. It has been correlated with several causes of mortality in adulthood: low birth weight with diabetes, nephropathy and cardiovascular diseases and high birth weight with autoimmune diseases and cancer. In genome-wide studies, an extended human leucocyte antigen (HLA) region has been linked to birth weight variation. We focused our attention on the HLA haplotypes marked by HLA-A, HLA-B and HLA-DRB1 polymorphisms in 1206 healthy Caucasian newborns belonging to the Cord Blood Bank of Pavia (Italy) and their mothers, aiming to investigate the association between this restricted HLA region and birth weight variation. In our study, the HLA-B*38;DRB1*13 haplotype showed an ascending trend among centiles addressing to the high foetal weight. The HLA-A*02;B*15 haplotype showed a descending trend among centiles addressing to the low foetal weight. Besides the acknowledged correlation between the HLA-A*02 and HLA-B*15 alleles (as well as low birth weight) and type I diabetes and between the HLA-B*38 and HLA-DRB1*13 alleles (as well as high birth weight) and several autoimmune diseases, we cannot predict if our babies, healthy at birth, will suffer from these pathologies during life. Nevertheless, our data point to the HLA telomeric end for markers linked to the low birth weight and to the HLA centromeric end for markers linked to the high birth weight, thus limiting the region involved in birth weight variation, which still represents a useful predictor of disease risk in adulthood.

Non classical HLA genes and non-HLA genes in a population of infants at familial risk of atopy.

AIM: We investigated on parental history and IgE serum level in 2588 consecutive newborns to individuate babies "at risk" of atopy at birth and we analysed the polymorphisms of class III region to evaluate the association with immunogenetic markers of HLA: C4A, C4B, LTA, RAGE and TNFA genes; we performed TNF and IgE receptor (FCERB1) physiologically related gene polymorphisms. RESULT: 791 babies/2588 (30.6%) were considered "at risk" for atopy and followed-up: 400 had familial history of atopy (at least one parent or sibling), 256 had IgE >0.35 kUA/l at birth and during the follow-up and 135 were positive for both conditions. The allele C4B2 was significantly more frequent in the sample of babies at risk (22.1% vs 10%, p< 0.001). Furthermore, the mean value of IgE at birth in babies carrying the allele C4B2 was 2.26 KUA/l versus 0.74 KUA/l in those not carrying this allele (p=0.01). No significant association emerged for RAGE at the centromeric end of class III region and for LTA, TNFA at the telomeric one. TNFRI, TNFRII and FCERB1 gene polymorphisms also seemed not implicated. CONCLUSION: Our study confirms that HLA class III region seems involved in familial predisposition to atopy, and C4B gene probably acts as a marker of a more restricted subregion.

A first study of cytokine genomic polymorphisms in CFS: Positive association of TNF-857 and IFNgamma 874 rare alleles.

OBJECTIVE: In the past two years we have developed a biological bank of genomic DNA, cDNA, serum and red blood cells of Italian patients with certified CFS from the two Italian referral centers for the syndrome. Recent studies have shown an imbalance in cytokine production in disease states similar to Chronic Fatigue Syndrome (CFS), such as sickness behavior, both in animals and in humans. However we notice that serum cytokine concentrations are often inconstant and degrade rapidly. With this in mind, we investigated cytokine gene polymorphisms in 80 Italian patients with CFS in order to ascertain whether in this group of patients it is possible to describe a genetic predisposition to an inflammatory response. METHODS: We analyzed the promoter polymorphisms of IL-10, IL-6 and the IFNgamma 874 T/A polymorphism in intron 1 with a PCR-SSP method (Cytogen One Lambda Inc. Canoga Park, CA, U.S.A) in 54 patients and TNF-308 G/A and -857 C/T promoter polymorphisms with a PCR-RFLP method (in 54 and 80 patients respectively). RESULTS: There is a highly significant increase of TNF -857 TT and CT genotypes (p = 0.002) among patients with respect to controls and a significant decrease of IFN gamma low producers (A/A) (p = 0.04) among patients with respect to controls. CONCLUSIONS: We hypothesize that CFS patients can have a genetic predisposition to an immunomodulatory response of an inflammatory nature probably secondary to one or more environmental insults of unknown nature.

Hierarchy of baby-linked immunogenetic risk factors in the vertical transmission of hepatitis C virus.

Mother-to-infant transmission of Hepatitis C Virus (HCV) represents the major cause of pediatric HCV infection today. Immunogenetic influence has been poorly investigated and mainly confined to HLA-class II serological polymorphisms. Among 290 parities, 135 from Pavia and 155 from Bergamo, of HCV-RNA-infected Italian women, 21 babies (7.24%) were HCV-RNA positive at birth and steadily positive over 20 months of life. All the 21 infected babies and 44 randomly selected uninfected ones, born to HCV-RNA+ mothers but steadily negative for HCV-RNA during a follow-up of 2 years, and their mothers were investigated for HLA-G, -C, -DRB1, -DQA1 and -DQB1 genomic polymorphisms. Among the different covariates, HLA-Cw*07, -G*010401, -DRB1*0701, -DRB1*1401 and homozygosity for HLA-G 14bp deletion can be considered as risk factors for HCV vertical transmission. On the contrary, protection was conferred by the HLA-DQB1*06, -G*0105N, -Cw*0602, DRB1*1104 and -DRB1*1302 alleles. Our initial question was: has the immunogenetic profile any role in the protection of the fetus growing in an infected milieu and, if so, is it independent from the other non-immunogenetic parameters? The answer to both questions should be yes.

Efficacy trial of pipobroman in polycythemia vera and incidence of acute leukemia.

A trial was conducted between 1970 and 1981 with pipobroman (PB) in 100 consecutive patients with polycythemia vera (PV), followed for a median time of 60 months, to evaluate the efficacy of this drug and assess the risk of acute leukemia. Phlebotomy was not done before PB was given. Hematologic remission was achieved in 92% of previously untreated patients in a median time of 12 weeks (range, 6-48 weeks) and maintained for a median of 48 months. Acute hematologic toxicity was absent. The median overall survival was 140 months with 65% five-year complication-free survival; the overall death rate at 12 years was 23% (6% of patients died of thrombotic complications). The actuarial risk of acute leukemia was 6% and 9% at five and seven years, and the time from the diagnosis of PV to leukemia ranged from 14 to 81 months. Myelofibrosis occurred in three patients and lung carcinoma in one. All leukemias were nonlymphoid with prominent monocytic component and dyserythropoiesis. One patient had erythroleukemia, two cases were heralded by preleukemia with chromosomal abnormalities, one involving the chromosomes 5 and 7. PB is effective in PV; however, despite an easy induction of remission, continuous low-dose maintenance is necessary and the risk of subsequent acute leukemia is still significant.

Factors influencing post-transfusional platelet increment in pediatric patients given hematopoietic stem cell transplantation.

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) always require platelet transfusions, but the increase in platelet count is often less than expected. Since factors responsible for poor response to platelet transfusions in this clinical setting are largely unknown, we performed a prospective study in 87 consecutive children transplanted in a single institution. The mean 16-h corrected count increment (CCI) of 598 platelet transfusions was 5.76 +/- 8.32 x 10(9)/l. Both before and after HSCT, 13.8% of patients had antibodies against HLA and/or platelet-specific antigens. Univariate analysis identified 12 factors significantly associated with a lower post-transfusion CCI, but only four reached statistical significance in the multivariate analysis. These four factors were concomitant therapy with vancomycin, alloimmunization, use of an Autopheresis cell separator for preparation of platelet concentrates and cytomegalovirus infection. We, therefore, suggest that a better response to platelet transfusions could be obtained by choosing a suitable cell separator, by avoiding the use of vancomycin and by adopting measures that reduce alloimmunization and CMV infection. Moreover, screening patients for HLA and platelet-specific antibodies before HSCT would identify the majority of subjects who will develop alloimmune refractoriness after transplantation and would allow the search for a compatible donor in advance.

Immunogenetic and hormonal study of cryptorchidism.

Ninety-four cryptorchids, 50 monolateral and 44 bilateral, aged from 2-9.4 yr (mean, 5.1 +/- 0.5 yr), were studied for the hormonal and immunogenetic profile. Pituitary-gonadal function was studied by evaluation of basal and peak GnRH-stimulated serum FSH and LH. In 83 cases, the serum testosterone (T) level was measured before and after CG treatment. No significant differences, between patients and age-matched controls, were found in either FSH or LH levels, whether under basal conditions or after GHRH stimulation. The mean basal serum T level was similar in mono and bilateral cryptorchids and in controls but, on the 15th day after treatment, it was significantly lower in the bilateral cryptorchids (P less than 0.05). CG administration led to testicular descent in 42 patients (23 with monolateral and 19 with bilateral cryptorchidism) and failed in 41 (21 with monolateral and 20 with bilateral cryptorchidism), independently of T increase. Immunogenetic investigation demonstrated that HLA-A11 and A23 were significantly overrepresented in the whole group of cryptorchids in comparison with the controls (P = 0.004 and P = 0.0123, respectively). HLA-A11 was more common in the bilateral form (P less than 0.05), whereas HLA-A29 was more frequent in the monolateral one (P less than 0.05). Forty percent of the bilateral cryptorchids with unsuccessful treatment had the HLA-A11 allele (P less than 0.01) and 70% the HLA-DR5.

Impact of leukapheresis cell composition on immunomagnetic cell selection with the Baxter Isolex 300i device: a statistical analysis.

Immunomagnetic cell selection (ICS) of CD34(+) cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34(+) ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34(+) cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34(+) cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34(+) cells (>500 x 10(6)) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

The morphologic spectrum of dilated cardiomyopathy and its relation to immune-response genes.

Endomyocardial biopsies from 174 patients with dilated cardiomyopathy (DC) were examined. Eight patients with histologically proven myocarditis were excluded from the study. A peculiar pattern of oversized and bizarre nuclei was observed in only some of the remaining patients. Two groups were identified: those with and without this feature (groups A and B, respectively). Myocyte width, nuclear diameter and nuclear/sarcoplasmic ratio were significantly higher in group A. The mean respective values were 36 +/- 5 mu, 14 +/- 3 mu and 0.41 +/- 0.08 for group A versus 20 +/- 8 mu, 7 +/- 2 mu and 0.37 +/- 0.08 for group B. Interstitial fibrosis was similarly present in groups A and B. Endocardial thickness was significantly increased in all patients, with group A showing the highest mean value. The morphologic features showed no correlation with the clinical condition of the patients at time of presentation. HLA typing was performed in 50 consecutive patients, 38 from group A and 12 from group B. DR4 and DR5 antigens were significantly more frequent in DC patients than in a normal population control (400 blood donors), while DR3 was less frequent. Group A was more strongly associated with the DR5 antigen than group B (55.3 vs 25.0%, respectively). It was less strongly associated with the DR4 antigen compared with group B (21.5 vs 41.7%, respectively). No difference was observed between the 2 groups concerning negative association with the DR3 antigen. Endomyocardial biopsies from DC patients reveal marked morphologic changes from patient to patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Low incidence of severe acute graft-versus-host disease in children given haematopoietic stem cell transplantation from unrelated donors prospectively matched for HLA class I and II alleles with high-resolution molecular typing.

We evaluated the outcome of 63 children given haematopoietic stem cell transplantation from unrelated donors (URD-HSCT) prospectively selected using DNA high-resolution typing of both HLA class I and class II loci. Thirty patient/donor pairs (48%) were fully matched. Among the others, HSCT was performed in the presence of one (n=22), two (n=9), or three (n=2) HLA disparities. Patients had either malignant (n=46) or non-malignant (n=17) disease. In all cases, graft-versus-host disease (GVHD) prophylaxis consisted of cyclospor-in A, short-term methotrexate and pretransplant anti-thymocyte globulin. The probability of haematopoietic recovery at day 100 was 97%. Two patients experienced primary graft failure. The cumulative probability of grades III-IV acute GVHD and of extensive chronic GVHD equalled 8 and 14%, respectively. A total of 12 patients died of transplant-related complications. The probability of transplant-related mortality (TRM) at 100 and 180 days was 10 and 15%, respectively, whereas the cumulative incidence of TRM was 22%. The probability of GVHD-related mortality equalled 6% at 2.5 years. The overall and disease-free survival rates were 67 and 65%, respectively. URD-HSCT with donor selection based on high-resolution HLA typing is associated with low incidence of both severe acute GVHD and graft failure. The observed outcome is comparable to that of children transplanted from HLA-identical siblings.

Successful T-cell-depleted, related haploidentical peripheral blood stem cell transplantation in a patient with Fanconi anaemia using a fludarabine-based preparative regimen without radiation.

Haematopoietic stem cell transplantation (HSCT) represents the treatment of choice for severe bone marrow failure in patients with Fanconi anaemia (FA). When the donor is a compatible relative, the chance of being cured with an allograft is in the order of 70%. However, for FA children lacking an HLA-identical sibling, the results of HSCT from an alternative donor are less satisfactory because of a higher risk of graft rejection, graft-versus-host-disease (GVHD) and regimen-related toxicity. We report on a 12-year-old girl with FA, who was treated by T-cell-depleted (TCD) peripheral blood HSCT from her haploidentical uncle, using a novel fludarabine-based preparative regimen without radiation. She had rapid engraftment with no toxicity and no GVHD. Progressive recovery of both numbers of lymphocyte and of proliferative response to polyclonal activators occurred over time. At 18 months after transplantation, she is well with 100% donor chimerism and has recovered normal immune function.

Chemotherapy and donor peripheral blood progenitor cells for acute leukemia in early relapse after allogeneic bone marrow transplantation.

Ten patients with acute leukemia (AL) in early relapse after allo-BMT were treated with a modified MEC (mitoxantrone, etoposide and Ara-C) regimen followed by donor PBPC collected after mobilization with G-CSF. Seven patients achieved CR or had normal hemopoietic reconstitution: two had an early relapse at days +53 and +48, two patients died from acute GVHD at days +31 and +96, one died of interstitial pneumonia at day +55, and two patients experienced long-term survival. One patient with refractory disease and nodal involvement who did not respond to the first BMT had overt expansion of the leukemia at day +36; one patient with Ph+ ALL and one with ANLL evolving from MDS, both with skin involvement, had blast cells in peripheral blood at day +27 and +26, respectively. Transient cytopenia occurred in all patients; a normal granulocyte and platelet count was achieved within 3 weeks in all patients but one; acute GVHD occurred in six patients, and four had chronic GVHD. This approach is feasible in patients in early relapse after allo-BMT. It assists prompt re-establishment of normal donor hematopoiesis avoiding the prolonged cytopenia observed after donor lymphocyte infusion in AL patients relapsed after allo-BMT.

A new method for placental/cord blood processing in the collection bag. I. Analysis of factors involved in red blood cell removal.

We describe a new procedure for large-scale CB processing in the collection bag, thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C, the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal, the cell pellet was resuspended and the percent recovery of total WBC, CD34+ progenitor cells, CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300, 600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC, CFU, CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy, effective and particularly suitable for large-scale banking under GMP conditions.

Multicyclic dose-intensive chemotherapy with circulating progenitor cell support for high-risk primary breast cancer.

The feasibility and safety of the administration of multiple cycles of dose-intensive chemotherapy (CT) supported with repeated reinfusions of circulating progenitor cells (CPCs) were evaluated in a prospective study of adjuvant initial therapy of poor-prognosis breast cancer. Eighteen patients with resectable breast cancer involving >/= 10 axillary nodes or >/= 5 axillary nodes and negativity of the estrogen receptor status received a cycle of standard FEC regimen (5-FU 600 mg/m2, epirubicin 60 mg/m2, CTX 600 mg/m2, i.v. on day 1) followed by G-CSF as CPC mobilization technique. Collected CPCs were fractionated and reinfused, with G-CSF, after each of the 4 subsequent cycles of high-dose FEC (HD-FEC) (5-FU 750 mg/m2, epirubicin 120 mg/m2, CTX 3 g/m2, i.v.) planned at 21 day intervals. The median numbers of CD34+ cells and CFU-GM collected (with one or two leukaphereses per patient) were 9.7x10(6)/kg (range: 2.5-22.9) and 9.9x10(4)/kg (range: 1.9-27.3), respectively, and day 9 was the median first day of procedure (range: 8-12) after FEC. All patients received the 4 courses of HD-FEC (for a total of 72 cycles). Hemopoietic recovery was rapid after each cycle and there was no treatment-related delays in CT administration. Mucositis was the major non-hematological toxicity. There were 2, 3, 7 and 9 episodes of WHO grade 3/4 mucositis in cycles 1, 2, 3 and 4, respectively. These severe episodes lasted a median of 4 days (range: 2-6) but no patient required parenteral nutrition. The mean +/- SD total hospital stay lasted 10 +/- 2 days. The delivery of 4 cycles of dose-intensive FEC CT supported by CPCs (mobilized with a single course of standard-dose FEC + G-CSF) is feasible and safe. It could represent an effective alternative strategy to other more aggressive programs for the adjuvant therapy of high-risk early breast cancer.

HLA and hypocomplementemia: the disadvantage of carrying the HLA-B35 and the silent alleles of the C4 complement component.

Hypocomplementemia is an extremely complex phenomenon: we devoted our attention to its immunogenetic basis, particularly to the HLA haplotypes involved and to the study of C4 polymorphic genes. With this in mind we analyzed a group of unrelated patients with hypocomplementemia and 15 families suffering from specific C4 deficiency. Firstly, we performed a population analysis in order to identify a statistically significant association: HLA-B35 and C4BQ0 alleles, in the total group of hypocomplementemic individuals, seem to be associated with the primary disease. Secondly, we defined HLA haplotypes clear-cut segregation in the hypocomplementemic families and we identified the most common HLA haplotypes carrying B35 and C4 null allele associated with this condition. With the aid of correspondence analysis and the Transmission Disequilibrium Test (TDT), we measured the strength of this association. In this work, mainly through family analysis, we envisaged a potentially interesting genomic trait, within HLA, close to B locus, that seems to be involved in hypocomplementemia itself and perhaps in hypocomplementemia-related disorders.